This research will develop a systematic way to use combined sum frequency generation (SFG), double resonance SFG (DRSFG), attenuated total refection FTIR (ATR-FTIR), and four-wave mixing (FWM) studies on alpha-helical structures to characterize membrane peptide/protein orientation in a single lipid bilayer in situ. The methodology can deduce the absolute orientation and complicated orientation distribution for a membrane peptide or protein in a single lipid bilayer that closely resembles the real membrane environment. It can also study the effect of asymmetry in lipid bilayers on peptide orientations, and can characterize structural information of peptides with very small surface coverage. The orientations of various antimicrobial peptides in lipid bilayers obtained in this study can help to elucidate modes of actions of such peptides on membranes. Antibiotic resistance is one of the most pressing problems in medicine at present, and we believe that this research will impact the design and optimization of peptides for antimicrobial purposes. The methodology will also be applied to study orientations of subunits of G-proteins in various environments, lending unique insight into how receptors and G proteins are organized in membranes during signal transduction and providing fundamental insights into various diseases such as cardiac failure. The specific aims are: 1. SFG studies supplemented by ATR-FTIR and FWM research can provide unique orientational information of various membrane peptides in a single lipid bilayer. These studies will lead to the determination of more detailed orientation distribution of the peptides in the membrane environment. The result here will also provide a calibration base for the studies proposed in Specific Aim 2. 2. DRSFG will be used to investigate the peptides examined in Specific Aim 1 to show that DRSFG can greatly improve the sensitivity of normal SFG. Unique structural information of membrane peptides with a very low surface concentration (peptide-lipid molar ratio<1:5,000) can be characterized using DRSFG. 3. In addition to ?-helical peptides, ?-helical structures in proteins will also be investigated to demonstrate the feasibility of determining structural information of secondary structural domains of membrane proteins and the orientation of membrane proteins using SFG, supplemented by ATR-FTIR and FWM. The G?1?2 subunit of a trimeric G-protein will be used as a model in this research. PUBLIC HEALTH RELEVANCE In this research a combination of vibrational spectroscopic techniques can provide vital orientational information regarding membrane peptides and proteins, which is difficult to obtain otherwise. Such work enables in-depth understanding of membrane orientations of antimicrobial peptides and G-proteins, providing important information to develop cures for infectious diseases, heart disease, asthma, opioid addiction, and hypertension.